CN114752661A - Application of PCK2 in preparation of medicine for preventing, relieving and/or treating osteoporosis - Google Patents

Application of PCK2 in preparation of medicine for preventing, relieving and/or treating osteoporosis Download PDF

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CN114752661A
CN114752661A CN202210429204.1A CN202210429204A CN114752661A CN 114752661 A CN114752661 A CN 114752661A CN 202210429204 A CN202210429204 A CN 202210429204A CN 114752661 A CN114752661 A CN 114752661A
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pck2
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bone
osteoporosis
lentivirus
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周永胜
李峥
张萍
刘云松
吕珑薇
张晓�
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Peking University School of Stomatology
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Abstract

The invention discloses application of mitochondrion phosphoenolpyruvate carboxykinase PCK2 in preventing, relieving and/or treating osteoporosis. The PCK2 gene is found to have the function of treating osteoporosis. Based on the effect of PCK2 in treating osteoporosis, the PCK2 can be used for preparing medicines for preventing, relieving and/or treating osteoporosis and/or other osteogenic dysfunctional diseases, or can be used as health products or nutritional supplements, so that the treatment of osteogenic dysfunctional diseases can be realized more conveniently and effectively at lower cost.

Description

Application of PCK2 in preparation of medicine for preventing, relieving and/or treating osteoporosis
The invention relates to the field of gene function and application, in particular to application of mitochondrion phosphoenolpyruvate carboxykinase (PCK 2) as a target gene in preparation of drugs for preventing, relieving and/or treating osteoporosis.
Background
Osteoporosis is a metabolic disease of bone in which bone mass is decreased, bone microarchitecture is degenerated, and bone fragility is increased, thereby easily causing fracture. Can occur at any age and become increasingly severe with age. According to the report of the world health organization, the number of osteoporosis patients in the world is more than two hundred million, which is the common frequently-occurring disease in the sixth place. Osteoporotic fractures are the most common and serious complications affecting osteoporotic patients and are characterized by difficulty in prevention, non-union after fracture or a significant increase in delayed union rate. With the coming and the accelerating progress of the aging of China society and the rising incidence rate of osteoporosis, a novel efficient anti-osteoporosis medicine needs to be developed.
The anti-osteoporosis medicine mainly comprises a bone resorption inhibitor, a bone formation promoter and bone mineralization. The bone resorption inhibitor mainly comprises estrogen, diphosphate, calcitonin, etc.; the bone formation promoter mainly comprises fluoride, parathyroid hormone and the like; the bone minerals mainly comprise calcium agent, vitamin D, etc. Estrogen can inhibit bone resorption, but acts on other organs of the whole body, increases the risk of breast and uterine cancer, and is not taken as a conventional medicament at present; the long-term application of the diphosphate can cause the reduction of bone strength and osteomyelitis of jaw bones and also can cause adverse reactions of digestive tracts; calcitonin can inhibit bone resorption and bone salt dissolution, and may cause adverse reactions such as flushing, nausea, and emesis of facial or body skin. Fluoride can promote osteoblasts to form new bone, but long-term application of the medicine can easily cause calcification defects; the lack of adequate evidence for long-term safety of parathyroid hormone can cause side effects such as headache, joint pain, etc. The pure adoption of bone minerals has no obvious curative effect on osteoporosis, and meanwhile, the long-term intake of a large amount of calcium can cause hypercalcemia, influence iron absorption, constipation and the like; vitamin D and its metabolite can promote intestinal absorption of calcium, participate in calcium and phosphorus regulation, promote calcification of skeleton, however, supplementing vitamin D excessively may lead to osteoclast activation and bone absorption increase, and its effect on bone mass is bidirectional. The long-term use of anti-osteoporosis drugs in patients with osteoporosis is limited due to a number of side effects. Therefore, the development of safer, more efficient and high-yield anti-osteoporosis drugs and gene therapy approaches is urgently needed.
Bone Marrow Mesenchymal Stem Cells (BMMSCs) have the potential of multidirectional differentiation, and promoting osteogenic differentiation of the BMMSCs is an effective method for treating bone metabolic diseases. Phosphoenolpyruvate Carboxykinase (PEPCK) is a key rate-limiting enzyme for gluconeogenesis in the liver and kidney, and plays an important role in cellular energy metabolism. Research shows that (mitochonddrial phospho-lateral kinase, PCK2) PCK2 can regulate osteogenic differentiation of mesenchymal stem cells through autophagy, but no literature reports on PCK2 and adipogenic differentiation exist at present, and no report on preparation of medicines for promoting osteogenesis and treating osteoporosis of PCK2 exists.
Disclosure of Invention
Aiming at the defects of the existing medicine for treating osteoporosis, the invention provides application of PCK2 in preparing the medicine for promoting osteogenesis. The invention firstly proves that the PCK2 has the potential of resisting osteoporosis in vivo, and provides a new idea for preventing and treating osteogenesis abnormal diseases, such as osteoporosis.
In order to solve the technical problems, according to the invention, an estrogen deficient and age-increasing osteoporosis mouse is taken as a test object, after the lentivirus is purified by injecting Pck2 into a rat tail vein of the osteoporosis mouse, osteogenic differentiation of mouse bone marrow mesenchymal cells (mBMMSCs) can be promoted, adipogenic differentiation of the mouse bone marrow mesenchymal cells can be inhibited, the bone density, the bone volume fraction and the thickness and the number of bone trabeculae of the estrogen deficient and age-increasing osteoporosis mouse can be increased, the ratio of the bone surface area to the bone volume and the separation degree of the bone trabeculae can be reduced, and thus the effects of treating the estrogen deficient and age-increasing osteoporosis can be achieved. The invention firstly proves that PCK2 inhibits the adipogenic differentiation capability of mBMMSCs by promoting the recovery of the osteogenic differentiation capability of mBMMSCs of osteoporosis mice, thereby reducing the bone loss.
Further research finds that the inhibition of the lipidogenic differentiation of the mesenchymal stem cells through autophagy is a key mechanism for reversing the osteoporosis expression of PCK 2. Based on the research, the invention provides application of the PCK2 gene serving as a drug target in screening drugs for preventing, relieving and/or treating osteoporosis. And the application of PCK2 in preparing medicines for preventing, relieving and/or treating osteoporosis.
The invention discovers a new function of the PCK2 gene, namely the PCK2 gene has the function of treating osteoporosis.
Based on the effect of PCK2 in treating osteoporosis diseases, the PCK2 can be used for preparing medicines for preventing, relieving and/or treating osteoporosis and/or other osteogenic dysfunctional diseases. The PCK2 is a key rate-limiting enzyme for gluconeogenesis and is a natural substance in cells, so that the PCK2 has a wide development and application prospect in treating estrogen-deficiency and age-increasing osteoporosis, and the pharmacological function is safer, the drug effect is milder, and the side effects are fewer.
Compared with the existing medicine for treating osteoporosis, the mitochondrial phosphoenolpyruvate carboxykinase PCK2 overexpression purified lentivirus for treating osteoporosis has the following advantages:
The mitochondrial phosphoenolpyruvate carboxykinase PCK2 in the product is a key rate-limiting enzyme for energy metabolism in human bodies, and can inhibit the in-vivo and in-vitro adipogenic differentiation of mesenchymal stem cells by regulating and controlling autophagy of cells. The over-expressed and purified lentivirus preparation PCK2 has safe source, high purity and titer, can be produced safely in large scale, increases in vivo energy metabolism enzyme by applying the preparation, can obviously improve bone density, thereby realizing the purpose of preventing and treating osteoporosis, and has wide development and application prospect in the medicine for preventing and treating the osteoporosis of human body. In addition, the purified lentiviral formulation of the present invention has no side effects or few side effects, and does not cause any adverse reactions in patients, and therefore can be effectively used for the long-term prevention or treatment of osteoporosis.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the results of construction of a mouse model for osteoporosis and detection of the expression level of Pck2 in mBMMSCs; a is a micro-CT detection graph of the metaphysis of the sham and OVX group femur ; b is a graph of H & E measurement results of the epiphyseal end of femur , C is a graph of results of fat measurement analysis in the H & E measurement results graph ([ P <0.05, [ P <0.01, [ P <0.001) ], D is a measurement result of protein expression amounts of Runx2, Pck2 and Ppar- γ in bmmscs ([ P <0.01, [ P <0.001) ], E is a graph of detection of micro-CT at the epiphyseal end of femur in the control group (6w) and the augmentation group (18 m); f is a graph of H & E assay results of the epiphyseal end of its femur , G is a graph of adipogenesis measurement assay results in the graph of H & E assay results (× P <0.05, × P <0.01, × P <0.001), H is a test result of the protein expression amounts of Runx2, Pck2 and Ppar- γ in bmmscs (× P <0.01, × P < 0.001);
FIG. 2 is a graph showing the results of testing the therapeutic effects of OVX group injected with Pck2 purified lentivirus; a is micro-CT of femur epiphysis, B is graph of H & E detection result of femur epiphysis, C is graph of measurement and analysis result of micro-CT (ns P >0.05, P <0.01, P <0.001), D is graph of serum ELISA quantification result (ns P >0.05, P < 0.05);
FIG. 3 is a graph of the results of a femoral adipogenesis assay with OVX group injected with Pck2 purified lentivirus; a is a graph of H & E measurements of the epiphyseal end of femur , B and C are graphs of the results of measurement and analysis of adipogenesis in the H & E measurements (ns P >0.05, P <0.01)
FIG. 4 is a graph showing the results of examining the therapeutic effects of aged mice injected with Pck2 purified lentivirus; a is micro-CT of femur epiphysis, B is graph of H & E detection result of femur epiphysis, C is graph of measurement and analysis result of micro-CT (ns P >0.05, P <0.01, P <0.001), D is graph of serum ELISA quantification result (ns P >0.05, P < 0.05);
FIG. 5 is a graph of the results of a femoral adipogenesis assay in aging mice injected with Pck2 purified lentivirus; a is a graph of H & E measurements of the epiphyseal end of femur , B and C are graphs of the results of measurement and analysis of adipogenesis in the H & E measurements (ns P >0.05, P <0.01)
FIG. 6 is a test of the effect of PCK2 in promoting adipogenic differentiation of hBMMSCs; a is a graph of oil red O staining and quantitative result after fat forming induction of hBMMSCs in a control group and a PCK2 knock-down group ([ P ] 0.05), B is a graph of detection result of mRNA relative expression of key fat forming indexes PPAR-gamma and C/EBP-alpha after fat forming induction of hBMMSCs in the control group and a PCK2 knock-down group ([ P ] 0.01), C is a graph of oil red O staining and quantitative result after fat forming induction of hBMMSCs in the control group and a PCK2 overexpression group ([ P ] 0.05), and D is a graph of detection result of mRNA relative expression of key fat forming indexes PPAR-gamma and C/EBP-alpha after fat forming induction of hBMMSCs in the control group and a PCK2 overexpression group ([ P ] P < 0.01); e is an H & E staining result graph (ruler is 100 mu m) of the subcutaneous ectopic adipogenic effect of the naked mice with hBMMSCs in the control group, the PCK2 knock-down group and the overexpression group, and F is an oil red O staining result graph (ruler is 100 mu m) of the subcutaneous ectopic adipogenic effect of the naked mice with hBMMSCs in the control group, the PCK2 knock-down group and the overexpression group;
FIG. 7 is a graph showing the detection of bone marrow mesenchymal stem cell autophagy in OVX group and 18m group osteoporotic mice; a is a detection graph of relative expression quantity of mRMA of a key osteogenic index Runx2 and Alp after osteogenesis induction of sham and OVX group mBMMSCs (nsP >0.05, P < 0.01); b is a protein expression quantity detection map of a key osteogenesis index Runx2 and a key cell autophagy index LC3B after osteogenesis induction of the sham and OVX group mBMMSCs; c is a detection graph of relative expression quantity of mRMA of adipogenic key indicators Ppar-gamma and C/ebp-alpha after adipogenic induction of sham and OVX group mBMMSCs (ns P >0.05, P < 0.01); d is a protein expression quantity detection map of a key indicator PPAR-gamma of adipogenesis after adipogenesis induction of the sham and OVX group mBMMSCs and a key indicator LC3B of cell autophagy; e is a detection graph of relative expression levels of mRMA of 6w groups and 18m groups of mBMMSCs after osteogenesis induction, namely Runx2 and Alp (ns P >0.05, P < 0.01); f is a protein expression quantity detection map of key osteogenesis indexes Runx2 and LC3B after osteogenesis induction of 6w groups and 18m groups of mBMMSCs; g is a detection graph of relative expression quantity of mRMA of fat forming key indicators Ppar-gamma and C/ebp-alpha after fat forming induction of 6w groups and 18m groups of mBMMSCs (ns P >0.05, P < 0.01); h is a protein expression quantity detection map of a key indicator of adipogenesis Ppar-gamma after adipogenesis induction and a key indicator of autophagy LC3B of mBMMSCs in 6w groups and 18m groups.
Detailed Description
The invention is further illustrated with reference to the following figures and examples.
Example 1 animal experiments demonstrated that PCK2 promotes the recovery of osteogenic differentiation ability of bone marrow mesenchymal cells in osteoporotic mice and inhibits their adipogenic differentiation
The experimental process comprises the following aspects:
1. animal models of mice in the group of ovarian castration (OVX) -induced osteoporosis and SHAM Surgery (SHAM) control groups were constructed.
(1) Test materials: sterile gauze, iodophor, 75% alcohol, 1% sodium pentobarbital, 1ml syringe, ophthalmological forceps, ophthalmological scissors, hemostatic forceps, spoon, 5-0 absorbable suture, needle holder and angle needle.
(2) Animal grouping: a total of 60C 57 mice 6-8 weeks old were randomly divided into 6 groups. The specific grouping is as follows: SHAM group + empty lentivirus tail vein injection group (SHAM + vector) 10; 10 patients in the SHAM + PCK2 lentivirus tail vein injection group (SHAM + PCK 2); the SHAM group + PCK2 lentivirus tail vein injection group +3-MA (SHAM + PCK2+3-MA) 10; ovariectomy + empty lentivirus tail vein injection (OVX + vector) 10; ovariectomy group + PCK2 overexpression lentivirus intraperitoneal injection group (OVX + PCK2) 10; ovariectomy group + PCK2 Lentiviral tail vein injection group +3-MA (SHAM + PCK2+3-MA)10 individuals. Performing a sham operation on groups 1-3, and performing an ovariectomy operation on groups 4-6.
(3) The operation process is as follows: after 1% pentobarbital is subjected to intraperitoneal injection anesthesia, abdominal skin and muscle layers are cut layer by layer, two ovaries are found, the ovaries are ligated on an oviduct, the ovaries are completely cut off, and the abdominal skin and the muscle layers are sewn in a layered and aligned mode. The SHAM group was operated as the OVX group except that tubal ligation and ovariectomy were not performed.
2. Mouse model for increasing osteoporosis
Animal grouping: a total of 30 18 month-old C57 mice were randomly divided into 3 groups as an aging mouse model and 30 6 week-old C57 mice were randomly divided into 3 groups as a control group. The specific grouping is as follows: 10 control + empty lentivirus tail vein injection (6w + vector); 10 of the control group + PCK2 lentivirus tail vein injection group (6w + PCK 2); 10 control groups + PCK2 lentivirus tail vein injection group +3-MA (6w + PCK2+ 3-MA); the age-increasing group and the unloaded lentivirus tail vein injection group (18m + vector) are 10; the age-increasing group + PCK2 overexpression lentivirus intraperitoneal injection group (18m + PCK2) comprises 10 animals; the age-increasing group + PCK2 lentivirus tail vein injection group +3-MA (18m + PCK2+3-MA) 10.
3, after modeling by OVX and SHAM for 3 months, taking femurs of 10 mice out of each group, and carrying out micro-CT scanning after fixing one femurs on one side for 24 hours by using 4% paraformaldehyde; 18m and 6w mice, femurs of 10 mice were taken out of each group, and one side of the femurs was fixed with 4% paraformaldehyde for 24 hours before micro-CT scanning. Micro-CT scanning using the Inveon MM system (Siemens), mouse femoral specimens were scanned for bone mass and microstructure, and three-dimensional reconstruction and data analysis were performed. Analyzing 0.5-1 mm of femoral metaphysis, including bone density (BMD), bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular spacing (Tb.Sp).
After Micro-CT scanning, decalcifying with 10% EDTA for 2 weeks, dehydrating, clearing, waxing, paraffin embedding, slicing (thickness of 5 μm), hematoxylin-eosin staining, H & E, and mounting.
Both Micro-CT analysis and H & E staining showed a significant decrease in the amount of femur epiphyseal bone in OVX mice, and successful construction of the estrogen-deficient model (see fig. 1A-1B). The femur epiphysis bone mass of 18m mice was significantly reduced, and the model of age-related osteoporosis was successfully constructed (see FIGS. 1A-1B).
3. Meanwhile, after the remaining femur was repeatedly washed with PBS containing 2% double antibody, both ends of the femur were cut off, and the α -MEM medium was aspirated with a 1ml syringe to sufficiently wash out the bmmscs, which was then cultured in vitro. The cell culture conditions were: 5% carbon dioxide, and culturing at 37 deg.C. The components of the cell Proliferation Medium (PM) are as follows: alpha-MEM + 20% (volume fraction) FBS + 2% penicillin-streptomycin double antibody, and after passage, alpha-MEM + 10% (volume fraction) FBS + 1% penicillin-streptomycin double antibody is replaced. The mBMMSCs used in the experiment are generation 2-3, the culture medium is replaced every 2 days, and the cells grow to 80-90% confluence state and then passage is carried out.
(1) The mRNA of mBMMSCs is collected to carry out qRT-PCR experiment, the total protein of mBMMSCs is collected to carry out WB experiment, the expression change of Pck2 is detected, and the result shows that in the mBMMSCs of OVX mice, the expression of Ppar-gamma is increased along with the reduction of Runx2 expression, and the mRNA expression of Pck2 is also obviously reduced (see figure 1C). The specific experimental steps are as follows:
Extraction of Total cellular RNA
a. The stock culture was discarded and the cells were washed 3 times with PBS.
b. 1ml Trizol TM reagent (6 well plate single well) was added, left for 5 minutes and transferred to a 1.5ml EP tube.
c. Add 200. mu.l chloroform, vortex vigorously and let stand at room temperature for 5 minutes until delamination.
d.4 ℃ and centrifugation at 12000g for 15 minutes.
e. The supernatant was pipetted into a new EP tube.
f. Adding isopropanol with the same volume, turning upside down, mixing well, and standing for 10 minutes.
Centrifuge at 12000g for 10 min at g.4 ℃.
h. The supernatant was discarded, 1ml of 75% (aqueous DEPC) ethanol was added, and the EP tube was washed by gently inverting it upside down.
i.4 ℃, 12000g, and 5 minutes of centrifugation.
j. And (5) repeating the steps (8) and (9).
k. Discarding supernatant, drying at room temperature, precipitating, adding appropriate amount of DEPC water, dissolving completely, and utilizing
The NanoDrop 8000 spectrophotometer measures the RNA concentration, and the RNA is subpackaged and stored at the temperature of 80 ℃.
Reverse transcription to synthesize cDNA
a. In a 10. mu.l reverse transcription reaction system, for example, the required RNA volume is calculated as:
required RNA volume (. mu.l) ═ 500(ng)/RNA concentration (ng/. mu.l)
b. Preparing a reverse transcription reaction solution according to the instruction of a Takara kit:
TABLE 1 reverse transcription reaction solution
Figure BDA0003611091580000061
Figure BDA0003611091580000071
c. The reverse transcription reaction conditions were as follows:
15 minutes at 37 DEG C
85 ℃ for 5 seconds
Maintaining at 4 deg.C
d. The resulting cDNA was reverse transcribed and stored at-20 ℃.
Real-time quantitative PCR reaction
a. Configuring a 10-microliter reaction system, setting three auxiliary holes for each sample and each gene, and specifically using reagents and using amounts as shown in the following table 2:
TABLE 2 reaction system reagents and amounts
Figure BDA0003611091580000072
The primer sequences are shown in Table 3 below:
TABLE 3 qRT-PCR primer sequences
Figure BDA0003611091580000073
Figure BDA0003611091580000081
Primer abbreviations:
pck2/PCK 2: mitochondrial phosphoenolpyruvate carboxykinase (mitochondrion pyruvate carboxykinase)
Alp/ALP: alkaline phosphatase (alkaline phosphatase)
Runx2/RUNX 2: run-related transcription factor 2 (run-related transcription factor 2)
Ppar- γ/PPAR- γ: peroxisome proliferator-activated receptor (PPAR-gamma)
C/EBP-alpha/C/EBP-alpha: recombinant human CCAAT enhancer binding protein-alpha (CCAAT/enhancer binding proteins alpha)
Gapdh/GAPDH: glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase)
The PCR reaction conditions are as follows: hot start at 95 ℃ for 10 min, denaturation at 95 ℃ for 30 sec, annealing extension at 60 ℃ for 1 min for 40 cycles.
c. Data were analyzed using the Δ Δ Ct method:
taking Gapdh as an internal reference, subtracting the Ct value of the target gene from the Ct value of the internal reference gene to obtain a delta Ct, subtracting the delta Ct of the control group from the delta Ct of the experimental group to obtain a delta Ct value, and performing formula 2- ΔΔCtThe expression level of the gene of interest was calculated relative to Gapdh.
(2) And (3) collecting proteins of mBMMSCs to perform a western blot experiment, and detecting the protein expression change of the PCK 2. The results showed that in the bmmscs of OVX mice, the PPAR- γ protein expression increased with the decrease in Runx2 protein expression, and the protein expression of PCK2 was significantly decreased (see fig. 1D). The specific experimental steps are as follows:
preparation of protein samples
a. Discard the medium, rinse 3 times with PBS, scrape the cells in a petri dish with a plastic spatula, and transfer to a 1.5ml EP tube.
b.4 ℃ and centrifugation at 1000rpm for 5 minutes.
c. Discarding the supernatant, adding a proper amount of RIPA lysate (adding a protease inhibitor and a phosphatase inhibitor in advance) into the cell sediment, re-suspending the cell sediment, sequentially carrying out ultrasonic disruption on the sample in an ice bath for 30 seconds (the power is 300W, ultrasonic treatment is carried out once every 3-5 seconds, the interval is 3s, and the time is 3 times in total), and then standing on ice for 20-30 minutes.
d.4 ℃ and centrifugation at 12000g for 30 minutes.
e. The supernatant was collected into a new EP tube and stored at-80 ℃.
Measurement of protein concentration
The protein lysate is subjected to protein quantification according to the product instruction of a BCA quantitative detection kit (Thermo company), and the steps are as follows:
a. bovine Serum Albumin (BSA) standards were diluted with Milli Q water to gradient concentrations of 2000. mu.g/ml, 1500. mu.g/ml, 1000. mu.g/ml, 750. mu.g/ml, 500. mu.g/ml, 250. mu.g/ml, 125. mu.g/ml, 25. mu.g/ml, 0. mu.g/ml, respectively.
b. Preparing a color development liquid: a, B solutions were prepared at a 50:1 ratio, calculated as 100. mu.l of working solution per well.
c. Each sample was prepared into three wells, 10. mu.l each, followed by addition of 100. mu.l of a developing solution, mixing, and incubation at 37 ℃ for 30 minutes.
d. The absorbance value at 570nm was measured using a microplate reader.
e. And drawing a standard curve by taking the BSA standard substance as a reference, and calculating the protein concentration of the sample to be detected according to the obtained standard curve and the OD value of the sample.
Preparing protein samples
After the protein concentration was determined, the volume of protein containing the appropriate mass was calculated and 1 × protein loading buffer was added and PBS filled to make the total volume of all samples the same. The total volume of each sample is 20-40 mul, after mixing uniformly, denaturation is carried out for 5 minutes at 99 ℃, and then a western blot experiment is carried out, or temporary storage is carried out at-80 ℃.
Electrophoresis
Separating gel with proper concentration is prepared according to the molecular weight of the target protein. The comb was removed and the protein sample was added. And (3) carrying out electrophoresis at a constant voltage of 60V until the concentrated gel runs out, and increasing the voltage to 100-110V until bromophenol blue runs out, so that the electrophoresis is stopped.
Rotary film
PVDF membrane with proper size is cut, soaked and activated for 30 seconds by anhydrous methanol. And (3) manufacturing a membrane conversion sandwich, paying attention to no air bubbles between the gel and the PVDF membrane, fixing a clamp, putting the membrane conversion sandwich into a membrane conversion groove, and paying attention to the correspondence of the positive electrode and the negative electrode. And (3) performing 100V constant-voltage electric conversion under an ice bath condition, and adjusting the film conversion time to be 40-90 minutes according to the molecular weight.
Blocking, antibody incubation
And (3) after membrane conversion, sealing the membrane for 1-2 hours at room temperature by using 5% skim milk sealing solution prepared by 1 xTBST.
Preparing an anti-dilution solution from the antibody dilution solution according to the antibody use instruction, putting the anti-dilution solution and the membrane into a hybridization bag together, and incubating overnight in a shaking table at 4 ℃.
TBST washes the membrane 3 times for 10 minutes each.
According to the following steps: 10000 ratio, diluting the secondary antibody with TBST, and incubating for 1 hour at room temperature.
Exposure method
TBST washes the membrane 3 times for 10 minutes each.
The two liquids in the chemical developing solution are mixed in equal volume according to the instruction, the PVDF membrane is incubated for about 3 minutes, and exposure is carried out by using a chemical developing machine.
4. Intravenous injection of mouse tail
(1) OVX mice were injected tail vein starting 3 months after surgery and aged mice at 18 months. Both the vector of the control group and the lentivirus titer purified by over-expressing Pck2 are 1X 108TU/mL, 1 injection, 200. mu.l total, sacrificed one month. Lentivirus was purchased from giga mart, su, and the cloning vector was LV 5. Performing Micro-CT and H&E, dyeing, wherein the method is the same as the step 1 (4).
The results showed no significant change in bone mass at the epiphyseal end of femur after injection of vector control lentivirus compared to mice in the group without OVX injection, whereas injection of Pck2 purified lentivirus group showed a significant increase in bone mass compared to the OVX group. This suggests that purified lentivirus Pck2 can significantly improve mouse osteoporosis (see FIGS. 2A-2B).
The results show that after injection of vector control lentivirus, there was no significant change in epiphyseal bone mass of femur compared to mice in the non-injected age-increasing (18m) group, whereas injection of Pck2 purified lentivirus significantly increased bone mass in the 18m group. This indicates that purified lentivirus Pck2 can significantly improve mouse osteoporosis (see FIGS. 2A-2B).
(2) After in vitro culture of mBMMSCs, osteogenic induction was performed. The specific method comprises the following steps: after the cells are converged to about 60%, discarding the original Proliferation Medium (PM), and gently washing the cells for 2-3 times by using a sterile PBS buffer solution; replacement of osteogenic induction medium (α -MEM + 10% (volume fraction) FBS + 1% celin-streptomycin diabody +10mM β -sodium glycerophosphate +0.2mM ascorbic acid +100nM dexamethasone, osteopenic medium, OM); the culture medium is replaced every 2 to 3 days.
(3) After in vitro culture of mBMMSCs, adipogenic induction was performed. The specific method comprises the following steps: after the cells are converged to about 60%, discarding the original Proliferation Medium (PM), and gently washing the cells for 2-3 times by using a sterile PBS buffer solution; replacing with lipid induction medium (OM); the culture medium is replaced every 2 to 3 days. The specific experimental steps are as follows:
in vitro lipogenesis differentiation potency assay
The cells were treated with 10 5The density of each well was inoculated into 6-well plates, and the culture was performed in the general proliferation culture (PM) or adipogenic induction culture (AM) or adipogenic induction culture. Oil red O staining and quantification were performed after 14 days of adipogenic induction culture.
1. Preparation of dye liquor
Oil red O stock solution: 0.5g of dry oil red O powder was weighed out and dissolved in 100ml of 100% isopropanol. After fully dissolving, storing at 4 ℃ in dark.
Oil red O working solution: before dyeing, taking oil red O storage liquid, and according to the storage liquid: distilled water 3: 2, and filtering by filter paper for dyeing.
2. Oil red O dyeing
The medium was discarded, washed gently with PBS 3 times, and fixed in 10% neutral formalin for 1 hour. Formalin was discarded, washed 3 times with PBS and rinsed with 60% isopropanol. And adding oil red O working solution after completely airing. Dyeing for about 15 minutes, observing the dyeing condition under a mirror at any time, sucking the dye liquor after fat drops are dyed, flushing with PBS to stop dyeing, and taking a picture under the mirror to retain a shadow.
3. Oil red O quantitation
Adding 1ml of 100% isopropanol into each well of a 6-well plate dyed by oil red O, dissolving for 10 minutes by gentle shaking, sucking 100 mu l of isopropanol into a 96-well plate, and measuring the absorbance under the wavelength of 500nm by using an enzyme-labeling instrument.
Example 2 in vitro and in vivo experiments prove that the autophagy is the key regulation mechanism for PCK2 to inhibit the adipogenic differentiation of the mesenchymal stem cells
1. Constructing a PCK2 stable knock-down and overexpression cell line (lentivirus is purchased from Jima, Suzhou), carrying out oil red O staining and quantitative analysis after fat forming induction, and detecting the expression changes of fat-forming related factors PPAR-gamma and C/EBP-alpha by qRT-PCR and western blot; the hBMMSCs of the control group, the knock-down group/the over-expression group and the collagen film are mixed evenly, implanted into the skin of the back of a nude mouse, the material is taken after 8 weeks, and the H & E staining is used for detecting the in-vivo adipogenesis effect. As a result, it was found that the in vivo and in vitro adipogenic differentiation ability of the cells was enhanced after knocking down PCK2 (see FIGS. 3B-3E), while the opposite was shown after overexpressing PCK2 (see FIGS. 3F-3I). The results show that PCK2 negatively controls the adipogenic differentiation of hBMMSCs.
The lentivirus transfection procedure was as follows:
(1) and when the cells are fused to 30-40%, discarding the original culture solution, gently washing the cells for 2-3 times by using sterile PBS buffer solution, and replacing the fresh culture medium.
(2) The lentiviral stock solution (100. mu.l for a 10cm dish) was removed from the-80 ℃ freezer, thawed on ice, and carefully added to the medium along with 10. mu.l polybrene.
(3) The medium was replaced with fresh medium after 24 hours.
(4) And (3) observing the fluorescence intensity under a fluorescence microscope after 3-4 days of transfection, and adding puromycin containing 5-10 ng/ml according to the fluorescence intensity to perform drug resistance screening.
2. Nude mouse back subcutaneous implantation experiment for detecting PCK2 effect on in vivo adipogenesis differentiation of hBMMSCs
hBMMSCs of the PCK2 knockdown group (sh-PCK2) and the control group (NC) and the PCK2 overexpression group (PCK2) and the control group (vector) were mixed with the collagen membrane scaffold material, respectively, and loaded into a cryopreservation tube. The inoculation amount of cells in each tube is 1 multiplied by 106The volume of the collagen film scaffold material is about 8X 2mm3。
Then 4 groups of cells (each group n is 5) are placed in a shaker at 37 ℃ to be shaken gently for 1h, and then are centrifuged for 5min at 150g, so that the cells are fully attached to the collagen membrane.
The operation is carried out in an SPF animal operating room, male BALB/C nu/nu nude mice of 5 weeks old are taken, the weight is about 20g, five mice in each group are anesthetized by 1 percent (mass fraction) of sodium pentobarbital, the back skin is disinfected, a longitudinal incision with the length of about 12mm is made on the central line of the back, subcutaneous implantation cavities of the back are separated towards two sides respectively, and a cell-collagen membrane mixture is implanted and recorded. And (5) aligning and sewing, breeding in cages, and taking materials after 6 weeks.
3. Bone marrow mesenchymal stem cells of sham and OVX groups and 6w and 18m groups of mice are extracted, and after in vitro culture of mBMMSCs, osteogenic induction and adipogenic induction are carried out. The specific experimental procedures are shown in example 1 (4). Western blot detects protein expression levels of autophagy key protein LC3B, osteogenesis related protein RUNX2 and adipogenesis related protein PPAR in a sham group, an OVX group, a 6w group and an 18m group, an overexpression group and an overexpression Pck2+3MA group, and clearly defines the function of PCK2 in regulating osteogenic and adipogenic differentiation through autophagy.
The result shows that the expression level of RUNX2 is obviously increased after vector control group lentivirus injection compared with the mice of a group without OVX injection, and the expression level of RUNX2 is obviously reduced in the group overexpressing Pck2+3MA compared with the OVX group overexpressing Pck2 by the vector control group lentivirus injection. After vector control lentivirus injection, the expression level of RUNX2 is obviously increased compared with that of mice in a group without 18m injection, and the expression level of RUNX2 is obviously reduced in a group overexpressing Pck2+3MA compared with that of a group 18m which the vector control lentivirus is injected to overexpress Pck 2.
After vector control lentivirus injection, the PPAR-gamma expression level is obviously reduced compared with the mice of a non-injection OVX group, and the PPAR-gamma expression level is obviously increased in the over-expression Pck2+3MA group compared with the OVX group which is injected with the control lentivirus over-expression Pck 2. After vector control lentivirus injection, the PPAR-gamma expression level is obviously reduced compared with that of a mouse not injected with the 18m group, and the PPAR-gamma expression level is obviously increased in a Pck2+3MA overexpression group compared with that of the 18m group injected with the control lentivirus overexpression Pck 2.
The results show that after vector control lentivirus injection, the expression level of LC3B is obviously increased compared with that of mice in a non-injection OVX group, and compared with that of an OVX group in which the control lentivirus is injected to over-express Pck2, the expression level of LC3B is obviously reduced in an over-expression Pck2+3MA group. After vector control lentivirus injection, the expression level of LC3B is obviously increased compared with that of 18m mice which are not injected, and compared with that of 18m mice which are injected with control lentivirus and over-express Pck2, the expression level of LC3B is obviously reduced in a Pck2+3MA over-expression group.
In conclusion, the autophagy is a key regulation link for promoting the osteogenic differentiation of the mesenchymal stem cells of the bone marrow by PCK2, so that the treatment of the osteogenic dysfunction disease is realized more conveniently and effectively by targeting and synthesizing an autophagy intervention target point in the follow-up process, and the cost is lower.

Claims (4)

  1. The application of PCK2 gene as a drug target in screening drugs for preventing, relieving and/or treating osteoporosis.
  2. Use of PCK2 in the manufacture of a medicament for the prevention, alleviation and/or treatment of osteoporosis.
  3. 3. The use of claim 1 or 2, wherein the PCK2 inhibits adipogenic differentiation of bone marrow mesenchymal stem cells by autophagy.
  4. 4. Use according to any of claims 1 or 2, characterized in that: the injection of the PCK2 overexpression purified lentivirus preparation can increase the bone density, the bone volume fraction, the trabecular bone thickness and the number of the estrogen-deficient and age-increasing osteoporosis of mice, reduce the ratio of the bone surface area to the bone volume and the trabecular bone separation degree, and achieve the effect of preventing and treating the estrogen-deficient and age-increasing osteoporosis.
CN202210429204.1A 2022-01-05 2022-04-22 Application of PCK2 in preparation of medicine for preventing, relieving and/or treating osteoporosis Pending CN114752661A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140271673A1 (en) * 2013-03-15 2014-09-18 Arturo Orellana Use of lxr antagonists for treatment of side effects of elevated glucocorticoid levels
CN107308450A (en) * 2016-04-27 2017-11-03 中国科学院上海生命科学研究院 Glycerokinase as the therapeutic targets of carbohydrate metabolism disturbance disease purposes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140271673A1 (en) * 2013-03-15 2014-09-18 Arturo Orellana Use of lxr antagonists for treatment of side effects of elevated glucocorticoid levels
CN107308450A (en) * 2016-04-27 2017-11-03 中国科学院上海生命科学研究院 Glycerokinase as the therapeutic targets of carbohydrate metabolism disturbance disease purposes

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Title
ZHENG LI等: "Mitochondrial Phosphoenolpyruvate Carboxykinase (PCK2) Maintains the Function of Bone Marrow Mesenchymal Stromal_stem Cells to Rescue Osteoporotic Phenotype Partly Through Autophagy Dependent Manner", RESEARCH SQUARE *

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